The unique region of the minor capsid viral protein 1, VP1u, of the Parvovoridae has a phospholipase A2 (PLA2) activity that is required for host cell infection. This PLA2 activity is utilized by virions to escape from the endo/lysosomal pathway en route to the nucleus for replication of the packaged ssDNA genome. The VP1u domain also plays a role in (I) tissue tropism and genome expression efficiency for non-pathogenic members and (II) diseases associated with pathogenic members. With the exception of members of the Erythroparvovirus genus for which the VP1u is located on the exterior capsid surface, the capsids of other Parvoviridae members must undergo structural re-arrangements to carry out this function because the VP1u is localized inside the assembled capsid and must be externalized for function. However, the VP1u structures are unknown and the structural calisthenics, mediated by pH, which must confer a functional state on the capsid surface, has not been characterized for any parvovirus member. The lack of structural elucidation in the context of the capsid is likely due to low copy number (~1 in 10) within the assembled capsid. The goal of this R21 proposal is to obtain preliminary data toward a comprehensive characterization of this essential VP1u domain for the type members of four Parvovirinae subfamily genera, bovine parvovirus (BPV) for Bocaparvovirus, adeno-associated virus serotype 2 (AAV2) for Dependoparvovirus, human parvovirus B19 for Erythroparvovirus, and minute virus of mice for Protoparvovirus, using a combination of biochemical, biophysical, and structural biology approaches. Following protein expression and purification, we will study the VP1u under the pH and Ca2+ conditions which are experienced by the parvoviruses in the endo/lysosomal pathway to become infectious. We will evaluate the enzymatic activity, including the pH optima and requirement for Ca2+, and determine the structure and dynamics of VP1u and its PLA2 enzyme. We will also determine if interaction with lipid membranes is a requirement for adoption of a functional state. The long term goal of this project is to use circular dichroism (CD), hydrogen deuterium exchange-mass spectrometry (HDX-MS), nuclear magnetic resonance (NMR) spectroscopy, and electron paramagnetic resonance to monitor VP1u structure and dynamic changes with/without lipid membranes, NMR and X-ray crystallography to determine atomic resolution structures, and CD, differential scanning calorimetry, differential scanning fluorimetry, and HDX-MS to evaluate stability. Negative-stain electron microscopy will examine lipid interactions. Overall Impact: Project outcomes will provide mechanism information on this essential and dynamic VP domain. Given the absolute requirement of the VP1u for infectivity, a comparative type member analysis, specifically 3D structure, including active site juxtaposition and flanking residues, and level of PLA2 activity at different pHs, could aid vector optimization and development of control strategies for non-pathogenic (e.g. AAV) and pathogenic members (e.g. BPV and B19), respectively.